Dihydrofolate reductase continues to be of considerable pharmacological, biochemical and now genetic interest because this enzyme appears to be the site of action of the powerful and widely used chemotherapeutic drug, methotrexate. Since many of the unusual properties of the animal enzymes appear to be related to a unique sulfhydryl group, our current studies are concerned with the structure (amino acid sequence) of the chicken and bovine liver enzymes and the reactivity of these sulfhydryl groups. Thus sequence studies revealed that in both enzymes the single -SH group is located in the N-terminal region of the protein. Treatment of the chicken liver enzyme with an excess of sodium tetrathionate not only resulted in the covalent attachment of one mole of thiosulfate to this single -SH group but also resulted in a 5 to 10-fold increase in activity over the native enzyme. This is the first case of an enzyme exhibiting activation rather than destruction upon treatment with this reagent. Although the beef liver enzyme is activated by organic mercurials as well as high concentrations of urea, no reaction is observed with tetrathionate.